Human herpesvirus 6 (HHV-6) is a recently discovered eukayotic virus for which infection of humans appears to be widespread. Understanding the biology of infection and the molecular mechanisms of viral replication and pathogenesis are long-term objectives of the proposed research. Identification and characterization of major antigens expressed in HHV-6 represents an important step toward realizing these objectives. The specific aims are to: 1) identify, physically map, and determine the DNA sequences of genes encoding major HHV-6 antigens, 2) develop monospecific antisera to allow determination of the biochemical characteristics of the protein products of these genes, and 3) investigate the functional roles played by such antigens during replication of the virus in vitro. The experimental design will include identification by Southern blotting of HHV-6 genes which code for antigens and are homologues to those of other herpesviruses, and construction of a library of recombinant gamma-gtll bacteriophage with HHV-6 genomic insertions for non-homologous genes. Recombinant bacteriophage will be screened with high-titer human antiserum. Southern blot analysis will be used to physically map the genes to the viral genome and the DNA sequence determined by the dideoxynucleotide method. For those genes with evident splicing, complementary DNAs (cDNAs) will be synthesized, mapped and sequenced. Fusion proteins expressed from recombinants will be purified and used to immunize rabbits to generate a bank of monospecific antisera specific for HHV-6 antigens. These probes will be used to characterize such antigens, with studies including determination of molecular weights, post-translational processing, and compartmentalization. Functional roles played by these antigens will be investigated by studies of mRNA and protein temporal expression to aid in antigen classification, and by studies of the role played by a viral glycoprotein during entry of host cells. Glycoprotein research will include adsorption assays, immunoelectron microscopy, and fluorescence dequenching studies. The research proposed will contribute to a better understanding of the relationship of these antigens to the complex processes involved in viral infection and replication.